Identification of mutations in herpes simplex virus envelope glycoproteins that enable or enhance vector retargeting to novel non-hsv receptors

ABSTRACT

In one embodiment, the invention provides an HSV vector comprising a mutant gB and/or a mutant gH glycoprotein, where the viral envelope further comprises a non-native ligand specific for a protein present on the surface of a predetermined cell type. In another embodiment, the invention provides an HSV vector comprising (a) a mutant gC and/or gD envelope glycoprotein which comprises a non-native ligand specific for a protein present on the surface of a predetermined cell type; and (b) a mutant envelope glycoprotein other than gD.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 15/137,953, filed Apr. 25, 2016, which is acontinuation of co-pending U.S. patent application Ser. No. 13/641,649,which is a U.S. National Phase of International Patent ApplicationPCT/US2011/032923, filed Apr. 18, 2011, which claims the benefit of U.S.Provisional Patent Application 61/325,137, filed Apr. 16, 2010, each ofwhich is incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant NumbersCA119298, NS40923, and DK044935 awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 2,809 Byte ASCII [Text] file named“723867 ST25.TXT,” created on Apr. 25, 2016.

BACKGROUND OF THE INVENTION

In recent years, the potential of viral vectors or geneticallyengineered viruses for the treatment of a variety of human diseases hasbeen a topic of intense study worldwide. Herpes simplex virus (HSV) isamong the most promising platforms for these purposes because of itsefficient entry and spread into a wide range of cell types and itsability to accommodate expression cassettes for multiple or very largeforeign genes that can provide therapeutic functions.

Targeting of HSV infection to specific cells for the delivery oftherapeutic products or lytic infection of cancer cells requires (i)elimination of the native ability of the virus to interact with itsentry receptors, mainly nectin-1 and HVEM, and (ii) the availability ofa mechanism to trigger the virus entry process in response to virusengagement of alternate receptors. The attachment and fusion steps ofHSV infection are mediated primarily by components of the viralenvelope, a membranous structure containing at least 10 glycoproteins(gB, gC, gD, gE, gG, gH, gI, gJ, gL, and gM) and four non-glycosylatedintegral membrane proteins (UL20, UL34, UL45, and UL49.5). Of theglycoproteins, gB, gD, gH, and gL are essential for wild type herpesviruses to infect their host cells, while the remainder are dispensablefor viral attachment or internalization. Prior to HSV-1 entry, virionsare adsorbed to the cell surface through binding of gC and gB, toexposed glycosaminoglycans on the cell membrane. The entry process isthen initiated by the interaction of gD with one of its cognatereceptors, such as herpesvirus entry mediator (HVEM) or nectin-1.Receptor binding results in a conformational change in gD triggeringactivation of gB and a fourth envelope glycoprotein, gH, as theeffectors of fusion between the viral envelope and cell membranes.

The virus can also infect cells by moving transcellularly, (e. g., atthe sites of gap junctions), a process referred to as lateral spread.The process of lateral spread to neighboring cells also involves theenvelope proteins; however different proteins appear to be essential foreach process. Thus, for example, while gE, and gI are not essential forprimary infection at the cell surface, removal of either of thesegreatly inhibits lateral spread.

Based on this understanding of the HSV-1 cell attachment and entryprocess, gC and gD have been modified to eliminate recognition of theirnatural receptors (“detargeting”) and insert a targeting element toprovide a novel interaction with specific receptors on the target cell(“retargeting”). Although these approaches have shown promising resultsin terms of ablation of virus entry through the natural receptors, theefficiency of retargeted entry has not been universally high, thuslimiting the practical application of these vectors. In fact, there hasbeen only one example in the literature of efficient HSV-1 retargeting(Menotti et al., J. Virol., 82(20), 10153-61 (2008); Menotti et al.,PNAS USA, 106(22) 9039-44 (2009)), and some attempts to take advantageof this design (replacement of residues 61-218 of gD with a single-chainantibody [scFv] against HER-2) to target the EGF receptor (EGFR) usingan EGFR-specific scFv, have been unsuccessful.

It is clear, therefore, that a methodology is needed to enhanceretargeted virus entry and spread, as such can reduce the effectivevirus dose and thereby increase safety.

BRIEF SUMMARY OF THE INVENTION

The invention provides modified HSV vectors that exhibit enhanced entryof cells, either through direct infection and/or lateral spread. In oneaspect, HSV vectors of the present invention can directly infect cellsthrough interaction with cell proteins other than typical mediators ofHSV infection (e.g., other than nectin-1, HVEM, heparansulfate/chondroitin sulfate proteoglycans. In another aspect, theinvention provides an HSV vector, such as comprising mutant gHglycoproteins, which HSV vector exhibits lateral spread in cellstypically resistant to HSV lateral spread, such as cells lacking gDreceptors.

In yet another aspect, the invention provides an HSV vector comprisingan envelope having one or more mutant envelope proteins, whereby saidHSV vector exhibits at least 25% increased rate-of-entry after 20minutes when assayed at either 30° C. or 37° C. in Vero cells afterfirst incubating at 4° C. relative to an HSV comprising a wild-type gBand/or gH protein.

In another aspect, the invention provides an HSV vector comprising amutant gB and/or a mutant gH glycoprotein, wherein the HSV comprisesmutations at two or more of group of residues consisting of gB:D285,gB:A549, gB: S668, gH:N753, and gH:A778, wherein said mutations arerelative to the sequence of HSV-1 strain KOS derivative K26GFP orGenBank Accession No. AF311740 or GenBank Accession No. X03896.

In a further aspect, the invention provides an HSV vector comprising anenvelope having one or more mutant envelope proteins other than gD orgC, whereby said HSV vector infects a cell via interaction of gD and/orgC with a cell surface protein other than or in addition to known gD orgC receptors such as nectin-1, HVEM, and heparan sulfate/chondroitinsulfate proteoglycans.

In still another aspect, the invention provides a viral stock comprisingan HSV vector as described herein.

In another aspect, the invention provides a DNA molecule encoding an HSVvector as described herein. In particular, the invention provides a DNAmolecule comprising a sequence of nucleic acids encoding a mutant gBglycoprotein having a mutation at one or more of the following residues:gB:D285, gB:A549, and/or gB:S668, wherein the mutation in gB is relativeto the sequence of HSV-1 strain KOS derivative K26GFP or GenBankAccession No. AF311740, as well as a DNA molecule comprising a sequenceof nucleic acids encoding a mutant gH glycoprotein having a mutation atone or more of the following residues: gH:N753 and/or gH:A778, whereinthe mutation in gH is relative to the sequence of HSV-1 strain KOSderivative K26GFP or GenBank Accession No. X03896.

The invention provides a method of increasing the efficiency of viralentry of a retargeted HSV vector comprising (a) retargeting the HSVvector by mutating a gC and/or gD envelope glycoprotein to comprise anon-native ligand specific for a cell surface receptor, and (b) mutatingan envelope glycoprotein other than gD such that the resulting vectorcan enter a cell via the interaction between said non-native ligand andthe cell surface receptor at least 10 times more efficiently than acontrol HSV vector comprising the non-native ligand of (a) but lackingthe mutated envelope glycoprotein of (b). Optionally, the gC and/or gDenvelope glycoprotein is impaired for binding to its natural receptor oris deleted.

In another aspect, the invention provides a method of killing a cancercell comprising contacting the cancer cell with an HSV vector asdescribed herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts plaque-forming activities per viral genome for isolates1-4, parent virus K26-gD:2/3NI, and control K26GFP on B78H1 cells andderivatives expressing intact HSV entry receptors (HVEM, B78/A;nectin-1, B78/C) or a debilitated receptor (mutant nectin-1, B78/TMC).Biological titers of the virus stocks in pfu/ml were determined on eachcell line by standard procedures. Genome titers of the stocks in gc(genome copies)/ml were determined by qPCR for the viral ICP47 gene.Biological titers were divided by genome titers and the mean values±SDfrom 3 determinations plotted on a logarithmic scale. *, <10-7.

FIG. 2A is a time course of virus entry into B78/C cells. Cells wereincubated with 200 pfu of K-gB:wt or K-gB:N/T at 4° C. for 30 min,washed thoroughly, incubated at 37 or 30° C. for the indicated times,and treated with acidic buffer. Cells were then incubated at 37° C.under methylcellulose-containing media for 3 days to allow plaqueformation. Plaques were counted and the mean values±SD from 3determinations plotted.

FIG. 2B is a time course of virus entry into Vero cells. Cells wereincubated with 200 pfu of K-gB:wt or K-gB:N/T at 4° C. for 30 min,washed thoroughly, incubated at 37 or 30° C. for the indicated times,and treated with acidic buffer. Cells were then incubated at 37° C.under methylcellulose-containing media for 3 days to allow plaqueformation. Plaques were counted and the mean values±SD from 3determinations plotted.

FIG. 3A and FIG. 3B is a time course of virus entry into Vero cellscomparing wild-type HSV-1 KOS with derivatives containing a mutant gB(gB:N/T) and/or gH allele (gH:N753K/A778V). Cells were incubated with500 pfu of each virus at 4° C. for 30 min, washed thoroughly, incubatedat (FIG. 3A) 37° C. or (FIG. 3B) 30° C. for the indicated times, andtreated with acidic buffer. Cells were then incubated at 37° C. undermethylcellulose-containing media for 2 days to allow plaque formation.Plaque numbers were counted and divided by the number of plaques formedby the same virus incubated for 2 h at 37° C. without acid treatment tocalculate % entry. Mean values±SD from triplicate samples were plotted.

FIG. 4A depicts virus yields over time in lysates versus media for Verocells infected at an MOI of 3 for 1 h followed by acid treatment andincubation at 37° C. with fresh media. Cell lysates and media werecollected at 4, 8, and 24 h post-infection (pi) and titered on Verocells. Viruses were wild-type HSV-1 KOS and derivative K-gH:KV carryingthe gH:N753K/A778V mutant allele.

FIG. 4B depicts virus yields over time in lysates versus media for B78/Ccells infected at an MOI of 3 for 1 h followed by acid treatment andincubation at 37° C. with fresh media. Cell lysates and media werecollected at 4, 8, and 24 h post-infection (pi) and titered on Verocells. Viruses were wild-type HSV-1 KOS and derivative K-gH:KV carryingthe gH:N753K/A778V mutant allele.

FIG. 5 provides schematic representation of detargeted and retargetedgD-mutant constructs. Abbreviations: SP, signal peptide; TM,transmembrane domain; CT, cytoplasmic tail; aa, amino acids.

FIG. 6 depicts a time course of virus entry into gD-complementing Verocells (VD60) as measured by plaque formation at 2 dpi. Cells wereincubated with 20,000 gc of wild-type KOS, K-gD:Δ224/38C-scEGFR, orK-gB:N/T-gD:Δ224/38C-scEGFR at 4° C. for 30 min, washed thoroughly,incubated at 37° C. for the indicated times, and treated with acidicbuffer. Cells were then incubated at 37° C. undermethylcellulose-containing media for 2 days to allow plaque formation.Plaques were counted and the mean values±SD from 3 determinationsplotted.

FIG. 7 depicts results of viability assays of cells infected withwild-type KOS, K-gD:Δ224/38C-scEGFR, or K-gB:N/T-gD:Δ224/38C-scEGFR.A549 or U87 cells were infected at 0.01-10 gc/cell for 3 or 6 days andcell viability relative to uninfected cells was determined by MTT assay.Data points are the means±SD of 6 replicates.

FIG. 8A depicts vector biodistribution results after in vivoadministration of KOS or K-gB:NT-gD:A224/38C-scEGFR. U87 flank tumors innude mice were allowed to grow to 700-1,000 mm3 in size, and 5×10⁸ gc ofKOS or K-gB:NT-gD:A224/38C-scEGFR were administered by tail-veininjection (n=2/virus). All animals were sacrificed 2 days later, andtumors and various organs were collected for DNA isolation and qPCR forthe viral ICP47 gene. Virus load in the different tissues was calculatedas gc/100 ng total DNA.

FIG. 8B depicts a plot of tumor size over time for U87 cells injectedinto the flanks of nude mice, with administration ofK-gB:NT-gD:A224/38C-scEGFR or PBS as a negative control when tumorvolumes reached 140 mm³ (day 0). ANOVA analysis showed that thedifference between the two groups was statistically significant(P<0.0001).

FIG. 9 provides a schematic representation of a gD mutant constructretargeted to CEA by an anti-CEA scFv insertion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the surprising discovery thatparticular changes in HSV envelope proteins such as gH and gB candramatically affect the kinetics of infection, such as efficiency ofinfection, rates of entry, and lateral spread of HSV among cells. In oneembodiment, the invention provides an HSV vector comprising mutant gHglycoproteins, whereby said HSV vector exhibits lateral, i.e.,“cell-to-cell” spread in cells lacking gD receptors.

The inventive vector can be an HSV-1 vector or an HSV-2 vector, butpreferably is an HSV-1 vector. The vector can be derived from awild-type HSV strain or from a laboratory strain (e.g., KOS) or mutantstrain. In this context, the vector can be said to be “derived from” astrain by virtue of the mutagenesis of the vector being described withreference to the strain.

Typically, the mutant entry protein within the inventive HSV vector is aglycoprotein involved with viral entry, such as gB, gH, and the mutantHSV vector can comprise mutated versions of both. However, the mutantentry protein can be any protein effecting entry of the HSV vector intocells. In preferred embodiments, the mutant entry protein is other thangD, although the HSV vector can additionally comprise a mutant gD, suchas containing a ligand or other desired mutation.

Preferred mutations of gB or gH glycoprotein for use in the inventiveHSV vector occur at one or more of the following residues: gB:D285,gB:A549, gB:S668, gH:N753, and gH:A778. More preferably, the inventiveHSV vector comprises mutations at both gB:D285 and gB:A549, at bothgH:N753 and gH:A778, and/or at each of gB: S668, gH:N753, and gH:A778.More preferably, the HSV vector contains two or more of such mutations(e.g., 3 or more, 4 or more), and the inventive HSV vector can comprisemutations in all five of these residues. A preferred HSV vector hasmutations at gB:285, gB; 549, gH:753, and gH:778.

The mutations are referred to herein relative to the codon (amino acid)numbering of the gD, gB, and gH genes of the HSV-1 strain KOS derivativeK26GFP. The sequences for gB and gH of K26GFP differ from the sequencesdisclosed in GenBank # AF311740 (incorporated herein by reference) forgB and GenBank # X03896 (incorporated herein by reference) for gH asreflected in the following table:

TABLE 1 Amino acid Nucleotide position AF311740 K26GFP position(s)AF311740 K26GFP gB 313 T S 938-939 ACG AGC 315 A T 943 GCC ACC 515 H R1,544 CAC CGC X03896 X03896 gH 12 I L 1,011 ATT CTT 110 P S 1,305 CCGTCG 127 T I 1,357 ACC ATC 138 S A 1,389 TCG GCG 150 A T 1,425 GCC ACC532 A A 2,573 GCT GCG 633 R R 2,876 CGT CGCHowever, K26GFP may contain additional differences in the region of thegene corresponding to nucleotides 2,079-2,102 of GenBank X03896. Thus,it will be understood that the sequence of either KOS derivative K26GFPor GenBank Accession No. AF311740 can serve as a reference sequence forthe gB mutations discussed herein. Also, the sequence of either KOSderivative K26GFP or GenBank Accession No. X03896 can serve as areference sequence for the gH mutations discussed herein. However, theinvention includes homologous mutations in gB and gH of any HSV strain.

Typically, the mutation of the entry protein for inclusion in theinventive HSV vector is a substitution mutation; however, the inventionis not limited to substitution mutants. Especially preferred mutant gBor gH glycoproteins for use in the inventive HSV vector are selectedfrom the group of substitution mutations consisting of gB:D285N,gB:A549T, gB:S668N, gH:N753K, gH:A778V. Preferably, the inventive HSVvector includes combinations of these substitutions (such as two or moreof such substitutions (e.g., 3 or more, 4 or more, or all)), with thegB:D285N/gB:A549T double mutant, the gH:N753K/gH:A778V double mutant,and the gB: S668N/gH:N753K/gH:A778V triple mutant being preferredembodiments. gB:D285N/gB:A549T/gH:N753K/gH:A778V is the most preferredcombination.

Efficiency of infection of a virus, such as an HSV vector of the presentinvention, reflects the number of viral particles required to infect ahost cell, i.e., to produce a plaque. Efficiency of infection can bemeasured by any method deemed suitable by one of ordinary skill in theart, such as those described in the Examples provided herein. See alsoUchida et al., J. Virol. 83: 2951-61 (2010). Efficiency of infection ofan HSV vector of the present invention can be expressed as a ratio ofplaques to total virus particles. In a preferred embodiment, the ratiois desirably as close to 1:1 (plaques to total virus particles) aspossible, with infection efficiency optimized by maximizing the presenceof active particles within a virus stock. For example, preferred vectorshave a 1:100, 1:10, 1:5, or 1:3 infection rate. One of ordinary skill inthe art will readily be able to consider the number or percentage ofactive particles as compared to total number of particles (“genome copy”or “gc” number). In other preferred embodiments, efficiency of infectionof a retargeted HSV vector of the present invention is greater than thatof a retargeted control HSV vector having a wild-type gB or gH protein.It will be understood that a sample of HSV vector used in testingefficiency of infection, whether a wild-type HSV or mutant HSV vectorsof the present invention, desirably includes both active and inactivevirus particles, and calculations of efficiency most appropriately areprepared based on numbers of active virus particles rather than on total(active and inactive) virus particles.

Certain HSV vectors in accordance with the present invention exhibitincreased rate-of-entry relative to a control vector. Rate-of-entryreflects the amount of time at which the virus becomes resistant toinactivation by acidic wash of the cells, and can be measured by anymethod known to one of ordinary skill in the art, such as thosedescribed in the Examples provided herein. See also Uchida et al., J.Virol. 83: 2951-61 (2010). Another method for determining rate of entryis measurement of ICP4 expression at 6-8 hours post-infection. Inparticular, rate-of-entry assays of the present HSV vectors aretypically carried out by incubating Vero cells with HSV vectors at 4° C.for 30 minutes, and then shifted to 30 or 37° C. for various intervals,such as 2, 3, 5, 10, 15, 20, 30, 40, 45, 50, 60, 120, or 180 minutes,followed by acidic wash. One of ordinary skill in the art willunderstand that rate-of-entry assays for other vectors will necessarilybe conducted in suitable cells having appropriate receptors. Anappropriate control vector is a retargeted HSV vector that has awild-type gB or gH protein. The resulting cultures are overlaid withmethylcellulose-containing media and incubated for an interval such as48 hours before counting plaques. In other preferred embodiments, rateof entry of a retargeted HSV vector of the present invention is greaterthan that of a retargeted control HSV vector having a wild-type gB or gHprotein. For example, the rate of entry can be increased by 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50% or more than 50% as compared withwild-type HSV. Exemplary vectors in accordance with the presentinvention, thus, exhibit at least 25% increased rate-of-entry after 20minutes when assayed at either 30° C. or 37° C. in Vero cells afterfirst incubating at 4° C. relative to a wild-type HSV.

HSV vectors of the present invention can enter cells either by directinfection or by lateral spread and, in some embodiments, the inventiveHSV can infect and/or spread to cells normally resistant to HSVinfection. In preferred embodiments, HSV vectors of the presentinvention are capable of both direct entry and lateral spread, althoughin some embodiments, the vectors may have similar capacity or increasedcapability for one type of entry (i.e., direct vs. lateral) as comparedto a wild type HSV, with similar or decreased capability for the othertype of entry.

The inventive HSV vector desirably is able to enter a cell, whether bylateral spread or otherwise, normally resistant to HSV entry. In certainembodiments, the inventive HSV vector can directly infect a cell viainteraction with nectin-2, nectin-3, nectin-4 or one or more othernon-HSV receptors. For example, an HSV vector can infect a cell viainteraction of gD, and preferably via interaction of gD and gC with acell surface protein other than or in addition to nectin-1, HVEM, andheparin sulfate/chondroitin sulfate proteoglycans. In such HSV vectors,the inventive HSV vectors can comprise a viral envelope having one ormore mutant envelope proteins other than gD or gC. In other embodiments,the inventive HSV vector exhibits lateral spread in cells lacking gDreceptors. Mutant forms of gH, for example as described herein, can beincorporated into such HSV to effect such enhanced lateral spreadability.

In addition to having the mutant entry protein or proteins, theinventive HSV also can include a non-native ligand specific for aprotein or other suitable binding site present at the surface of apredetermined cell type. To contact the cell, the ligand preferably isattached to the surface of the HSV virion, such as by incorporation intoa viral envelope protein or glycoprotein (such as gC, gD, and the like).This can be achieved by expression as a recombinant fusion protein, forexample a fusion with a HSV surface protein or glycoprotein containingthe ligand, or by chemical crosslinking of the ligand to the virion orby the establishment of high-affinity biochemical interaction of thevirus envelope with the ligand, for example mediated throughbiotin-avidin binding.

The ligand can be any suitable agent that binds the surface of thepredetermined cell. The ligand typically is proteinaceous and canconstitute a natural binding partner for a cell surface protein (e.g.,EGF), a portion of an antibody (e.g., a single chain antibody (scFv), asingle domain antibody (VHH), or other ligand), or other binding agent.Where the predetermined cell is a cancer cell, a ligand can target aprotein present on the cancer cell. For example, the cancer cell candisplay a receptor such as EGFR, EGFRvIII, CEA, andClC-3/annexin-2/MMP-2, and the ligand can target such a receptor, i.e.,the ligand can be capable of specifically binding such protein. The cellcan be any contemplated cancer cell, although in preferred embodiments,the cancer cell is a lung epithelial carcinoma cell, a colonadenocarcinoma cell, a pancreatic adenocarcinoma cell, a glioblastomacell, an astroglioma cell, a vulvar epithelial carcinoma cell, or abreast carcinoma cell. Preferably, the cancer cell is in a mammal, suchas a human.

Replacement of a portion of gD (such as of residues 61-218) with a scFvtargeting a receptor such as HER-2 (also referred to as neu or erbB-2)can serve as a ligand for targeting certain cancer cells (thoseoverexpressing HER-2). Similarly, a scFv or other ligand, binding to thetumor-specific marker carcino-embryonic antigen (CEA) or thetumor-associated EGFR can be employed and insertion sites in gD can bebetween residues 1 and 25 or between residues 24 and 25. Furtherinformation concerning the ligand, as well as engineering HSV vectorscontaining ligands, is discussed in international patent publication WO1999/006583, the disclosure of which is incorporated herein byreference. When expressed as a fusion with an envelope protein, scFvs orVHHs are generally preferred over other types of ligands. scFvs and VHHSthat exclusively recognize mutant versions of the EGFR, such as aninternally deleted version called EGFRvIII, are preferred targetingligands. EGFRvIII and other mutant EGFR versions are specificallyexpressed on cancer cells and not on normal cells. EGFRvIII-specificantibodies, scFvs and VHHs have been described in the literature (Kuanet al, Int. J. Cancer, 88, 962-69 (2000); Wickstrand et al., CancerRes., 55(14):3140-8 (1995); Omidfar et al., Tumor Biology, 25:296-305(2004)).

In addition to having the mutant entry protein and the ligand, theinventive HSV vector can be further modified from a wild-type HSV. Forexample, in some embodiments, the inventive HSV vector can be used as anoncolytic virus. For such application, the HSV vector genome can bemodified similarly as HSV vectors currently under investigation asoncolytic vectors. Also, the genome of the inventive vector can beengineered to contain microRNA target sequences, such as miR21, miR124,and/or miR128, which can be employed to achieve preferential HSVreplication in tumor cells (see Lee et al., Clin. Cancer Res., 15(16),5126-35 (2009); Edge et al., Mol. Ther., 16(8), 1437-43 (2008); Caewoodet al., Plos Pathogens, 5(5), e1000440 (2009)). In this respect, controlof virus replication by cellular microRNAs can be achieved by insertionof microRNA target sequences into untranslated regions of essentialviral genes. MicroRNA recognition of the targeted viral mRNA causesdegradation of that viral mRNA (or blocks its translation). Thus thevirus will not be produced in normal cells that contain the regulatorymicroRNA, but will be produced in (e.g.) tumor cells that do not containthe microRNA. Alternatively, the genome can be rendered replicationincompetent and engineered to express one or more transgenes (see, e.g.,U.S. Pat. Nos. 5,804,413 and 7,531,167, which are incorporated herein byreference), which can encode proteins or polypeptides orbiologically-active RNAs (such as microRNA, interfering RNA, etc.).Accordingly, genome of the inventive HSV vector, whetherreplication-competent (oncolytic) or replication-defective, can compriseone or more exogenous expression cassettes (i.e., containingencoding-sequences in operable linkage with promoters, enhancers, andother suitable regulatory elements), such as encoding a transgeneexpressing marker (such as green fluorescent protein), an agent thatenhances tumor killing activity (such as TRAIL or TNF), or othertherapeutically-important gene product.

Further, the inventive HSV vector can have one or more viral envelopeglycoproteins impaired for binding to its natural receptor. In someembodiments, one or more viral envelope glycoproteins can be deletedaltogether. In preferred embodiments, the viral envelope glycoproteinthat is impaired or deleted is gC or gD.

The inventive HSV vector can be made by any suitable method, which areknown to those of ordinary skill in the art. Typically, the inventiveHSV vector will be constructed using recombinant DNA technology, wherebya gene encoding the mutant entry protein replaces the correspondingwild-type (or source) copy of the entry protein gene. Accordingly, HSVvectors according to the invention having a mutant gB and/or gH proteinhave a gene encoding the mutant gB and/or gH protein, respectively, andlack a gene encoding wild-type gB and/or gH, respectively.

To facilitate the manufacture of the inventive HSV vector, the inventionprovides a DNA molecule comprising a sequence of nucleic acids encodinga mutant entry protein suitable for inclusion into the inventive HSVvector. For example, the DNA molecule can encode any mutant gBglycoprotein described herein, such as having a mutation at one or moreof the following residues: gB:D285, gB:A549, and/or gB:S668 (such asgB:D285N, gB:A549T, and/or gB:S668N). Similarly, the DNA molecule canencode a mutant gH glycoprotein as described herein, such as having asequence of nucleic acids encoding a mutant gH glycoprotein having amutation at one or more of the following residues: gH:N753 and/orgH:A778 (such as gH:N753K and/or gH:A778V).

The DNA molecule can be in any suitable form, such as a plasmid, cosmid,or other construct. The DNA molecule can also include other sequencessuitable for propagation (ori sites), expression (e.g., promoters,enhancers, IRES sites and other regulatory sequences) or engineering(e.g., cassettes encoding toxins, markers or tags, restriction enzymerecognition sites, etc.).

The genetic constructs, and the HSV vectors, of the present inventioncan be constructed using standard techniques. For example, a relativelynew technique is manipulation of the HSV genome in bacteria as bacterialartificial chromosomes (BACs) (see, e.g., Gierash et al., J. Virol.Meth., 135, 197-206 (2006)). That the entire HSV genome is published(see, e.g., GenBank # X14112 (strain 17), and portions from otherstrains are similarly published (e.g., GenBank # AF311740 for gB and #X03896 for gH), further facilitates the construction of the inventiveHSV vectors and genetic constructs.

Generally, the inventive HSV vector is most useful when enough of thevirus can be delivered to a cell population to ensure that the cells areconfronted with a suitable number of viruses. Thus, the presentinvention provides a stock, preferably a homogeneous stock, comprisingthe inventive HSV vector. The preparation and analysis of HSV stocks iswell known in the art. For example, a viral stock can be manufactured inroller bottles containing cells transduced with the HSV vector. Theviral stock can then be purified on a continuous nycodenze gradient, andaliquotted and stored until needed. Viral stocks vary considerably intiter, depending largely on viral genotype and the protocol and celllines used to prepare them. Preferably, such a stock has a viral titerof at least about 10⁵ plaque-forming units (pfu), such as at least about10⁶ pfu or even more preferably at least about 10⁷ pfu. In still morepreferred embodiments, the titer can be at least about 10⁸ pfu, or atleast about 10⁹ pfu, and high titer stocks of at least about 10¹⁰ pfu orat least about 10¹¹ pfu are most preferred. Such titers are establishedusing cells that express the targeted receptor.

The invention additionally provides a composition comprising the HSVvector and a carrier, preferably a physiologically-acceptable carrier.The carrier of the composition can be any suitable carrier for thevector. The carrier typically will be liquid, but also can be solid, ora combination of liquid and solid components. The carrier desirably is apharmaceutically acceptable (e.g., a physiologically orpharmacologically acceptable) carrier (e.g., excipient or diluent).Pharmaceutically acceptable carriers are well known and are readilyavailable. The choice of carrier will be determined, at least in part,by the particular vector and the particular method used to administerthe composition. The composition can further comprise any other suitablecomponents, especially for enhancing the stability of the compositionand/or its end-use. Accordingly, there is a wide variety of suitableformulations of the composition of the invention. The followingformulations and methods are merely exemplary and are in no waylimiting.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

In addition, the composition can comprise additional therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. Factors thatcontrol inflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the vector and physiological distress. Immune systemsuppressors can be administered with the composition method to reduceany immune response to the vector itself or associated with a disorder.Alternatively, immune enhancers can be included in the composition toupregulate the body's natural defenses against disease. Antibiotics,i.e., microbicides and fungicides, can be present to reduce the risk ofinfection associated with gene transfer procedures and other disorders.

HSV vectors and compositions as described herein can be used in methodsof killing a cancer cell. In such methods, an HSV vector or compositionas described herein is applied to a cancer cell that has been removedfrom or is present in an organism, such as a mouse, rat, rabbit, cat,dog, pig, cow, chicken, monkey, or human, using methods known to one ofordinary skill in the art. In some exemplary embodiments, the cancercell is a lung epithelial carcinoma cell, a colon adenocarcinoma cell, apancreatic adenocarcinoma cell, a glioblastoma cell, an astrogliomacell, a vulvar epithelial carcinoma cell, or a breast carcinoma cell.

For treating cancer cells in vivo, a preferred embodiment of theinventive HSV comprises mutations in gB and/or gH as described herein,whereby efficient entry of the vector into tumor cells is achieved.Furthermore, such vectors additionally desirably comprises a targetingligand as described to alter the HSV tropism to target cancer cellspreferentially. The use of a cancer-specific ligand can facilitatetreatment of disseminated cancer and systemic delivery of the HSV,although for treating solid tumors, intratumoral delivery, such asstereotactic injection, can be employed. For example, Examples 13 and 14reveal preferential tumor targeting using EGFR- and CEA-specificligands, but other tumor antigens can be similarly employed. Such an HSVfurther can comprise microRNA target sequences to facilitatepreferential replication in cancer/tumor cells. The HSV thus can home totumor/cancer cells preferentially, minimizing infection of non-targetedcells, enter the tumor/cancer cells efficiently, and preferentiallyreplicate in tumor/cancer cells as opposed to healthy cells.Furthermore, such an HSV can be engineered to express ananti-cancer/tumor factor in tumors to further effect killing of thecancerous/tumor cells in vivo. Additionally, a replication-defective HSVcan be engineered to target other types of cells to efficiently andcell-specifically deliver therapeutic genes for other diseases.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example describes the identification of a hyperactive gB-mutant,gB:D285N/A549T (“gB:N/T”) through use of various cell lines modified forreceptivity to HSV infection.

Baby hamster kidney J1.1-2 and murine melanoma B78H1 cells (as describedin J Virol 83, 2951-2961) have been shown to be resistant to HSVinfection due to the absence of gD receptors (J Virol 72, 9992-10002; JVirol 76, 2424-2433; Mol Ther 3, 160-168). A mutant version of nectin-1,QN76-77AA/M85F, that is severely impaired for binding to gD and thusfails to support HSV entry, was previously described by Struyf et al. (JVirol 76, 12940-12950) and is referred to here as TMC (Triply MutatedHveC). Clonal TMC-expressing J and B78 cell lines were created by stabletransfection and designated J/TMC and B78/TMC. J/TMC and B78/TMC cellswere established by transfection of J1.1-2 or B78H1 cells with plasmidpcDNA3TMC and selection for resistance to 0.4 mg/ml or 0.8 mg/ml G418,respectively. The TMC expression plasmid pcDNA3TMC was created byreplacement of a V-domain-encoding fragment of pBG38 (Science 280,1618-1620) with the corresponding fragment of pTMC153-his (J Virol 80,138-148). Clonal lines obtained by limiting dilution or cylinder cloningmethods were confirmed for expression of the introduced receptor cDNAsin >95% of the cells by indirect immunofluorescence.

J/TMCΔC cells were established by transfection of J1.1-2 cells withplasmid pcDNA3TMCΔC and selection with 0.4 mg/ml G418. PlasmidpcDNA3TMCΔC, encoding TMC deleted for both C-domains, was created bydeleting the coding sequences for nectin-1 codons 148 to 336 frompcDNA3TMC.

The virus mutant K26-gD:R222N/F223I (J Virol 83, 2951-2961; Virology360, 477-491), abbreviated here as K26-gD:2/3NI, is a mutant of K26GFP(J Virol 72, 7563-7568) and has a highly diminished ability to usenectin-1 for infection due to a pair of mutations in gD, but is largelyunimpaired for infection through HVEM; it expresses a VP26-GFP fusionprotein (J Virol 72, 7563-7568) facilitating the detection of virusinfection and growth. K26-gD:2/3NI was challenged for growth on J/TMC orB78/TMC cells by reiterative high-MOI infection and progenyamplification on HVEM-expressing J (J/A) cells.

Two separate selections were carried out. In the first, J/TMC cells wereinoculated with K26-gD:2/3NI at 1,000 pfu/cell and rinsed after 8 h withacidic buffer (Table 1, Exp 1). Progeny virus harvested 2 d later fromthe cells and medium was expanded on J/A cells for infection of J/TMCcells at 100 pfu/cell, followed by acid treatment at 8 h. Progeny viruswas harvested as before and used for plaque purification by limitingdilution on J/TMC cells. The second selection (Table 2, Exp 2) wasperformed in a similar manner with the following modifications. B78/TMCcells were inoculated with the same initial virus at 100 pfu/cellfollowed by expansion on J/A cells, progeny virus was passaged twicemore on B78/TMC cells and once on J/TMCΔC cells. Plaque purificationswere performed on B78/TMC cells.

Following the above selections, virus was isolated from a number ofindividual J/TMC and B78/TMC plaques, purified, and characterized.Direct sequencing was used to identify mutations in the gD gene of sevenisolates derived from the first experiment and eight from the second. Asshown in Table 1, 10 of the 15 isolates had one or two new missensemutations in the gD ORF in addition to the parental 2/3NI mutations.Distinct amino acid substitutions were found in isolates from the first(A185T) and second selection (Q178H), and one isolate contained bothsubstitutions. However, five of the seven isolates from the firstexperiment harbored no new gD mutations, suggesting that these viruseshad undergone alterations outside the gD gene.

TABLE 1 gD mutations in selected virus isolates^(a). Substitution Group(parental + new)^(b) New base change^(b) Frequency^(c) Isolate #^(d) Exp1 (2 passages) 1 R222N/F223I — 5/7 A 2 R222N/F223I + A185T GCC→ACC 2/7 BExp 2 (4 passages) 3 R222N/F223I + Q178H CAG→CAT 7/8 C 4 R222N/F223I +Q178H/A185T CAG/GCC→CAT/ACC 1/8 D ^(a)K26-gD:2/3NI was passaged twice orfour times through TMC-expressing cells and progeny viruses were clonedby limiting dilution on J/A cells. ^(b)Amino acid or nucleotide changesin individual isolates. ^(c)Number of isolates with the indicatedmutation/total number of analyzed isolates in each experiment.^(d)Designation of representative isolate from each of the groups.

One representative isolate from each of the four gD-mutant groups(Table 1) was purified by repeated limiting dilution on J/A cells. Theisolates were amplified on J/A cells and their infection profilesestablished on B78 cells expressing HVEM (B78/A), nectin-1 (B78/C), TMC,or no gD receptor. Infections were performed with equal numbers of viralgenome copies (gc) determined by real-time quantitative PCR (J Virol 76,12940-12950). Control infections included K26-gD:2/3NI and its wild-typegD parent, K26GFP (J Virol 72, 7563-7568). Cells were infected for 8 hand expression of the tegument protein VP16 was visualized by indirectimmunofluorescence rather than GFP fluorescence in these assays becausethe intensity of the GFP signal varied among the isolates and theparental viruses.

Neither K26GFP nor the restricted K26-gD:2/3NI virus showed infection ofB78H1 cells even at 1,000 gc/cell, consistent with the absence of gDreceptors on these cells. In agreement with previous findings (J Virol83, 2951-2961), K26-gD:2/3NI showed substantially reduced infection ofB78/C, but not B78/A cells compared to K26GFP. Infection of B78/TMCcells by K26GFP was observed only at the highest gc input, whileinfection by K26-gD:2/3NI was undetectable, thus validating the use ofthis virus-receptor combination for the selection of gain-of-functionmutants. Isolate A showed a similar level of infection on B78/A cells asK26GFP and K26-gD:2/3NI. However, although this isolate had the same gDORF as K26-gD:2/3NI, it infected B78/C cells with an efficiency similarto that of wild-type gD virus rather than at the much lower levelobserved with K26-gD:2/3NI. This unanticipated result indicated thatother virus glycoproteins involved in the entry process had beenaltered. On B78/TMC cells, isolate A showed a dramatic increase ininfectious activity over its parent, as expected, but also greateractivity than K26GFP. Surprisingly, unlike K26GFP and K26-gD:2/3NI,isolate A was capable of infecting unmodified B78H1 cells, suggestingthe acquisition of mutations that render infection independent of knowngD receptors. ICP4 staining at 6 h post-infection (hpi) showedessentially the same trend, indicating that the changes in isolate Aprincipally facilitated virus entry rather than replication.

To quantify these results and identify potential differences betweenisolate A and the gD-altered isolates B-D, the plaque-forming activitiesof the four isolates and the two control viruses on the panel of B78cell lines were compared. The results of triplicate titrations of eachvirus on each cell line, expressed as plaque-forming units per genomecopy (pfu/gc) (FIG. 1), mirrored those of the single-round infectionassays for isolate A. Thus, all four isolates showed increased plaqueformation on B78/C cells compared to their K26-gD:2/3NI parent virus.Unlike the parent virus, all four formed plaques on B78/TMC and B78H1cells. In addition, the shared A185T substitution in gD of isolates Band D appeared to have a general enhancing effect on plaque formation,consistent with a previous report that A185T increases the efficiency ofviral cell-to-cell spread in a gD receptor-independent manner (J Virol74, 11437-11446).

Together, these results indicated that the four isolates had similarmutations outside the gD gene and that the identified mutations in gDwere not the primary cause of the acquired ability of these viruses togrow on B78/TMC and B78H1 cells. Furthermore, it is noteworthy that thespecific infectious activities (pfu/gc) of the four isolates were verysimilar on B78/TMC and B78H1 cells, indicating that the impaired gDreceptor TMC did not play a key role in the original selection of theseisolates.

Direct sequencing revealed that each of the four isolates harbored thesame two missense mutations in the gB ORF: D285N and A549T. The gH andgL ORFs of isolate A, were also sequenced, with the exception of anextremely GC-rich 20-nucleotide portion of gH (positions 1983-2102 inGenBank accession number X03896). No changes were found in either gene.

These results show that the double missense mutation in gB, referred tohereafter as N/T, was most likely responsible for the acquiredphenotypes of the selected isolates.

Example 2

This example characterizes functional changes caused by the N/T mutationas they relate to the infectious properties of the virus, particularlyviral entry via proteins other than nectin-1.

To confirm that new gD mutations in isolates B-D were not responsiblefor the extended tropism of these viruses, the cloned gD gene of eachisolate was expressed and evaluated to determine whether incorporationof its product into a gD-null virus would mimic the changes in host-cellrange observed with the complete isolate. The results of these transientcomplementation assays, conducted as described in J Virol 74, 2481-2487,showed that none of the newly acquired mutations in the gD genes ofisolates B-D substantially altered virus infection of any of the fourcell lines. Notably, the mutant gD alleles yielded no detectableinfection of B78/TMC cells although two of them were isolated followingselection on these cells. Furthermore, the acquired mutations showedminimal suppression on B78/C cells of the nectin-1-specific defectcaused by the parental 2/3NI mutations. These results supported thesuggestion that the gain-of-function phenotypes of the differentisolates were principally the result of changes outside the gD ORF.

Next, to evaluate the effects of the N/T mutations, a gB-null virus, KΔT(J Virol 61, 714-721), was rescued by homologous recombination withwild-type and N/T-mutant gB plasmids and plaque purification onnon-complementing Vero cells (African green monkey kidney cells). Verocells were co-transfected with KΔT (J Virol 61, 714-721) viral DNA andplasmids pgB1:wt or pgB1:D285N/A549T, followed by plaque purificationthrough three rounds of limiting dilution on Vero cells. Isolatesdesignated K-gB:wt and K-gB:N/T (K-gB:N/T), respectively, were confirmedby DNA sequencing through the entire gB and gD ORFs. Plasmid pgB1:wtcontains the gB ORF and flanking regulatory sequences from K26GFP (JVirol 72, 7563-7568); mutant counterparts, including pgB1:D285N/A549T,were created by replacement of appropriate fragments of pgB1:wt with thecorresponding fragments of PCR products generated on DNA from selectedvirus isolates.

Because gB contributes to virus attachment to cells by binding toglycosaminoglycans (“GAGs”) on the cell surface, gD receptor-deficientCHO-K1 cells and their GAG-deficient derivative pgsA-745 cells (ProtNatl Acad Sci USA 82, 3197-3201) were used to determine whether enhancedinfection by gB:N/T was dependent on GAG binding. At 8 hpi, K-gB:N/Tproduced readily detectable infection of CHO-K1 cells at a virus inputof 100 or more gc/cell, whereas no infection by K-gB:wt was seen at a10-fold higher dose and limited infection at a 100-fold higher dose. At24 hpi, K-gB:N/T produced foci of infected CHO-K1 cells, indicative ofdirect cell-to-cell spread, while a 100-fold higher dose of K-gB:wtyielded only individual infected cells and very small foci. OnGAG-deficient pgsA-745 cells, infection by K-gB:wt was barely detectableeven at 24 hpi at the highest virus input. In contrast, infected cellswere distinguishable at a 100-fold lower dose of K-gB:N/T at both 8 and24 hpi. Together, these results demonstrated that gB:N/T enablesinfection of gD receptor-negative cells and indicated that this effectwas not due to increased binding of gB to GAGs. Using a direct assay forvirion attachment, gB:N/T mutations did not enhance GAG-dependent orGAG-independent virus adsorption to cells.

Given the absence of known gD receptors on CHO-K1 cells (Cell 87,427-436), K-gB:N/T was analyzed to determine whether gD is required forthe enhanced infection of these cells. A gD-null virus was derived fromK-gB:N/T, designated K-gB:N/TΔgD, by replacement of the complete gD ORFwith that of EGFP. CHO-K1 cells were infected with 1,000 gc/cell ofK-gB:N/T or K-gB:N/TΔgD and stained for VP16 at 16 hpi. The resultsshowed that gD is indispensable for infection of CHO-K1 cells byK-gB:N/T. The requirement for gD in K-gB:N/T infection of CHO-K1 cellsraised the possibility that these cells express minor or cryptic gDreceptors on their surface that can serve as HSV-1 entry receptorsconditional to the presence of the gB:N/T double mutation. Nectin-3 wasconsidered as a candidate based on a previous report by Cocchi et al.that nectin-3 can mediate entry of HSV harboring a particularcombination of gD mutations (J Virol 78, 4720-4729). To perform aninfection blocking assay, CHO-K1 cells were incubated with ratanti-mouse nectin-3 (Cell Sciences) or rat anti-mouse nectin-4 (R&DSystems) mAbs (both IgG2a) or PBS for 1 h at RT, and then infected withK-gB:N/T at 3,000 gc/cell for 2 h at 37° C. followed by acid treatment.Infections were assessed at 16 hpi as described above. The resultsshowed that anti-nectin-3, but not isotype-matched anti-nectin-4,reduced infection by K-gB:N/T in a dose-dependent manner; phase-contrastimages indicated that this was not due to anti-nectin-3-mediated celldetachment. These observations suggested that nectin-3 plays anessential role in gB:N/T mutant virus infection of CHO-K1 cells, mostlikely by functioning as a receptor for gD.

Indirect immunofluorescence was performed as described previously (JVirol 83, 2951-2961), using goat anti-mouse nectin-3 or nectin-4polyclonal antibodies (R&D Systems) (1 μg/ml) as primary antibodies andCy3-conjugated rabbit anti-goat IgG (Sigma) (1:400) as secondaryantibody. Immunofluorescence analysis demonstrated the presence ofnectin-3 on the surface of CHO-K1 cells.

Since nectin-3 appeared to enable K-gB:N/T infection of CHO-K1 cells,other nectin-family members were evaluated for this function. Increasedinfection by both K-gB:wt and K-gB:N/T was seen on CHO-K1 cells thatoverexpress human nectin-2 (CHO/Nec2) (Virology 246, 179-189) ornectin-4 (CHO/Nec4) compared to unmodified CHO-K1 cells, indicating thatthese nectins can act as HSV entry receptors, but infection wasapproximately 100-fold higher in each case for K-gB:N/T than forK-gB:wt; neither virus infected any of these cell lines as efficientlyas they infected CHO-K1 cells expressing human nectin-1 (CHO/C (Science280, 1618-1620)). These data indicated that gB:N/T facilitates the useof multiple members of the nectin family for viral entry, suggestingthat the gB mutations act in a general manner to enhance virus infectionthrough weak gD-receptor interactions. A similar effect was seen in acomparison of K-gB:wt and K-gB:N/T infection of B78/TMC cells where thegD binding-impaired TMC mutant of nectin-1 represented the weakreceptor.

Example 3

This example demonstrates accelerated viral rate of entry by the gB:N/Tmutant allele.

Since mutations in gB have previously been shown to alter the kineticsof viral entry (J Virol 63, 730-738; Virology 122, 411-423; Virology137, 185-190), rate-of-entry assays were performed to determine whetherthe gB:N/T mutations might act in this manner. Nectin-1-expressing cells(B78/C) were used for these experiments because these cells aresusceptible to both the wild-type and the mutant virus after 24 h ofvirus/cell co-incubation However, under those conditions these cellsshowed no clear difference in infection efficiencies between K-gB:wt andK-gB:N/T. B78/C cells were incubated with K-gB:wt or K-gB:N/T at 200 pfuper well at 4° C., washed thoroughly, incubated at 37° C. or 30° C. for0-60 min, and extracellular virus was inactivated by low-pH treatment.The cells were then overlain with methylcellulose-containing media andincubated at 37° C. for 3 days to allow plaque formation. At 37° C.,entry of gB:wt virus progressed steadily over time whereas entry of themutant virus in the first 3 min exceeded that of the wild-type virus in60 min (FIG. 2A). However, following this initial phase of rapid entryby the mutant virus, additional entry was limited such that at 60 minnearly equal numbers of the two viruses had entered the cells. Thisobservation indicated that the dramatic differences at the early timepoints reflected a difference in virus entry kinetics rather than invirus input. At 30° C., entry also proceeded more rapidly for K-gB:NITthan for K-gB:wt although the extent of entry was lower overall than at37° C. These results demonstrated that the gB:N/T mutations acceleratevirus entry via the natural gD receptor nectin-1.

HSV-1 entry into gD receptor-transduced B78 cells reportedly takes placeby a low-pH-independent endocytic pathway (J Virol 79, 6655-6663),whereas entry into Vero cells occurs by membrane fusion at the cellsurface (Proc Nat'l Acad Sci USA 84, 5454-5458; J Virol 63, 3435-3443);entry into receptor-transduced CHO-K1 cells is mediated by alow-pH-dependent endocytic pathway (J Virol 77, 5324-5332; J Virol 78,7508-7517). To determine whether the gB:N/T mutations accelerate entrythrough each of these different pathways, the rate-of-entry assays wererepeated on Vero and CHO/C cells. As shown in FIG. 2B, the results onVero cells at 37° C. were similar to those on B78/C cells although bothviruses showed a delay in early kinetics compared to B78/C cells. As onB78/C cells, the entry kinetics were slower at 30° C., but the 60 minentry level of K-gB:N/T was as high as the maximum reached at 37° C.Since CHO/C cells do not form well-defined plaques, the rates of entryon these cells were assessed by anti-VP16 staining at 8 h post acidicwash to visualize infected cells. B78/C cells were included forcomparison. The entry kinetics of both viruses were similar on the twocell lines and consistent for B78/C cells with the results of FIG. 2A.K-gB:wt showed a gradual increase in infection, reaching a maximum at30-60 min, while as little as 3 min sufficed for near-maximum infectionby K-gB:N/T. These results demonstrated that the gB:N/T mutationsaccelerate virus entry into receptor-bearing cells regardless of thepathway used by wild-type virus.

Transiently complemented gB-null virus was used to determine whether theindividual mutations of gB:N/T affected the rate of virus entry. Verocells were transfected with expression plasmids for gB:N/T, gB:D285N,and gB:A549T, the cells were infected the next day with KΔT, andsupernatants harvested the following day were used for rate-of-entryassays on B78/C and Vero cells.

Rate-of-entry assays were performed as described previously (J Virol 63,730-738) with modifications. Cells were incubated with viruses at 4° C.for 30 or 60 min and washed three times with cold PBS. The cells werethen shifted to 37 or 30° C. for various intervals followed by acidtreatment. The cultures were incubated at 37° C. and stained for VP16expression at 8 h (primary infection) or 48-72 h (plaque formation).

The results of this assay showed that entry was accelerated on both celllines by either of the single mutant gBs compared to wild-type gB. TheA549T version of gB displayed a somewhat greater effect than the D285Nversion, and the double mutant gB mediated slightly more entry in 10 minthan either of the single mutants; these differences were clearer onVero cells than on B78/C cells which was not surprising given theobservation that the majority of K-gB:N/T virus entry into B78/C cellsoccurred in the first few minutes (FIG. 2A) while entry into Vero cellstook place at a slower pace (FIG. 2B). These results indicated that eachof the gB:N/T mutations contributed, albeit unequally, to the phenotypeof the double mutant protein.

Example 4

This example demonstrates enhancement of retargeted HSV infection by thegB:N/T mutant allele.

HSV infection has been retargeted by ablation of the nativereceptor-recognition functions of gD and insertion of recognitionelements for novel receptors (Proc Natl Acad Sci USA 103, 5508-5513; JVirol 82, 10153-10161; Proc Natl Acad Sci USA 106, 9039-9044). However,the efficiency of these retargeted infections has been reported to belower than that of natural infection through authentic receptors. Todetermine whether efficiency was improved over retargeted HSVscontaining gB:wt, gB:N/T was analyzed for its effect on targetedreceptor-dependent and -independent (“off-target”) infection. A gD-nullderivative of K-gB:wt, designated K-gB:wtΔgD, was generated byreplacement of the gD ORF with that of EGFP as done earlier to produceK-gB:N/TΔgD. Thus, K-gB:wtΔgD and K-gB:N/TΔgD were produced byco-transfection of VD60 cells with plasmid pΔgD-EGFP and viral DNAs ofK-gB:wt or K-gB:N/T, respectively, with subsequent purification ofgreen-fluorescent plaques on VD60 cells.

Using equal amounts of these two gD-null viruses (pfu determined ongD-complementing VD60 cells as described in J Virol 62, 1486-1494),transient complementation assays were performed with a retargeted gDconstruct, pgD:3C/Δ711/38C-scEGFR, containing mutations that severelyimpair virus infection through nectin-1 (A3C/Y38C) (J Virol 79,1282-1295, 2005; J Virol 83, 2951-2961, 2009) and HVEM (deletion ofresidues 7-11), and an insertion specifying a single-chain antibody(scFv) directed against the epidermal growth factor receptor (EGFR)between residues 24 and 25. The retargeting plasmidpgD:3C/Δ711/38C-scEGFR was generated by insertion of the 528 scFvsequence (Clin Cancer Res 12, 4036-4042) into plasmid pgD:3C/Δ711/38C-NE(0.1 Virol 79, 1282-1295).

The retargeted gD construct enabled infection exclusively ofEGFR-transduced CHO-K1 cells (CHO/EGFR) by K-gB:N/TΔgD but notK-gB:wtΔgD, while the parental wild-type gD construct complemented bothviruses for infection of CHO-K1 cells expressing the natural HSVreceptors HVEM (CHO/A cells) or nectin-1 (CHO/C), but not for infectionof CHO/EGFR cells.

As described earlier, entry of the gB:N/T virus into nectin-1-bearingcells was markedly accelerated compared to wild-type virus (K-gB:wt),suggesting that the gB:N/T double mutation affects a rate-limiting stepin entry. The gB:N/T double mutation was combined with anEGFR-retargeted gD allele, gD:Δ224/38C-scEGFR, in a wild-type virusbackground. The resulting virus entered EGFR-transduced J1.1-2 cells(J/EGFR) that lack authentic HSV receptors approximately 100-fold moreefficiently than the same virus lacking the gB double mutation.Furthermore, the double recombinant virus entered EGFR-transduced J1.1-2cells at least 10,000-fold more efficiently than J1.1-2 cells transducedwith the natural entry receptors, HVEM or nectin-1. Thus the gB doublemutation increased the efficiency of retargeted infection withoutyielding significant “off-target” infection through the natural HSVentry receptors. In addition, the double recombinant virus entered anumber of tumor cell lines expressing EGFR with similar efficiencies aswild-type virus entering these lines via the natural receptors. On mostof these cell lines as well, the retargeted virus lacking the gB doublemutation showed approximately 100-fold less entry.

These results strongly suggested that gB:N/T can augment targetedreceptor-dependent HSV infection without detectably increasingoff-target infection and hence, that these mutations may provebeneficial for the efficient targeting of therapeutic HSV vectors.

Example 5

This example provides a genetic selection approach to identifyadditional virus mutations to increase infection. Different from above,this selection uses the highly impaired gD receptor TMCΔC derived fromnectin-1 in combination with a wild-type gD virus instead of a mutant gDvirus.

As described in Example 1, J/TMCΔC cells are gD receptor-deficientJ1.1-2 baby hamster kidney cells that stably express a severelydebilitated version of the HSV entry receptor nectin-1, and which areused as a target for selection of complementing mutations. The defectivereceptor, J/TMCΔC, has mutations in the nectin-1 variable (V) domainthat reduce gD binding, and lacks the two constant (C) domains ofnectin-1. Inoculation of J/TMCΔC cells with K26GFP, a recombinant virusthat expresses GFP as a fusion with VP26, yielded no green fluorescenceeven at an MOI of 1,000. To confirm that this was due to the absence offunctional entry receptors, the cells were inoculated with areplication-defective HSV mutant, QOZHG, that expresses lacZ from theICP0 IE promoter and GFP from the CMV IE promoter, and virus entry wasassessed by X-gal staining at 24 hpi. No entry was detected on J/TMCΔCcells, while almost 100% entry was observed at the same virus input(MOI=10) on J cells expressing wild-type nectin-1 (J/C). The same resultwas obtained by observation of GFP signals. These findings indicatedthat the J/TMCΔC protein lacked any ability to function as an HSV entryreceptor and thus could be a suitable target for the selection ofcomplementing mutations.

J/TMCΔC cells (twenty 10-cm dishes) were inoculated with K26GFP at anapproximate MOI of 1,000 and rinsed with 0.1 M glycine (pH 3.0)(referred to hereafter as acidic wash) at 24 h post-infection (pi).Combined intracellular and extracellular virus harvested at 72 hpi(first-round product) was expanded on J/A cells for a second round ofinfection of J/TMCΔC (twenty 10-cm dishes) at an MOI of approximately1,000 and acidic wash at 24 hpi. Progeny virus was again harvested andexpanded (second-round product). After two more rounds of selection atthe same MOI and one round at an MOI of ˜300, plaques were purified bylimiting dilution on B78/TMC cells expressing full-length TMC. Selectedisolates were analyzed by selective sequencing. All mutant sequencesreported here were unambiguous, confirming the purity of the isolatesand the absence of wild-type virus.

To identify genetic alterations responsible for the ability of thefifth-round products to enter and spread on J/TMCΔC cells, 46 viruseswere individually purified by limiting dilution and propagated for DNAextraction. Surprisingly, direct sequencing of the gD ORFs of theseisolates demonstrated that only 16 (Nos. 31-46 in Table 2) harbored amissense mutation in this gene, while the remaining 30 (Nos. 1-30)showed no amino-acid changes in this region. Among the 16 isolates withsubstitutions in gD, 12 had A185T (Nos. 31-42), 3 had S140K (Nos.43-45), and 1 had S276L (No. 46). A185T and a different substitution atposition 140 (S140N) have been described previously (10, 41).

TABLE 2 Mutations in selected virus isolates^(a) No.^(b) gB^(d) gH^(e)gL^(e) No.^(b) gD^(c) gB^(d) gH^(e) gL^(e) 1 wt wt N753K/A778V wt 24 wt2 wt S 668N N753K/A778V* 25 wt 3 wt Hinc(−) N753K/A778V* 26 wt 4 wtHinc(+) N753K/A778V* 27 wt 5 wt Hinc(+) 28 wt 6 wt Hinc(+) 29 wt 7 wtHinc(+) 30 wt 8 wt Hinc(+) 31 A185T wt N753K/A778V* 9 wt Hinc(+) 32A185T S668N A571T 10 wt Hinc(+) 33 A185T Hinc(−) 11 wt Hinc(+) 34 A185THinc(−) 12 wt Hinc(+) 35 A185T Hinc(−) 13 wt Hinc(+) 36 A185T Hinc(−) 14wt Hinc(+) 37 A185T Hinc(+) 15 wt Hinc(+) 38 A185T Hinc(+) 16 wt 39A185T Hinc(+) 17 wt 40 A185T Hinc(+) 18 wt 41 A185T Hinc(+) 19 wt 42A185T Hinc(+) 20 wt 43 S140K Hinc(+) 21 wt 44 S140K Hinc(+) N753K/A778V*22 wt 45 S140K 23 wt 46 S276L Hinc(+) N753K1A778V* K26GFP was passagedfive times through J/TMCΔC cells and progeny viruses wereplaque-purified. ^(b)Isolate numbers referred to in the text. ^(c)Directsequencing was performed on the entire gD ORF. Amino acid substitutionsare indicated. wt, no substitution. ^(d)Results of direct sequencing ofthe complete gB ORF or diagnostic HincII digestion of PCR amplicons.Amino acid substitutions are indicated. wt, no substitution. Hinc(−),absence of the diagnostic HincII site. Hinc(+), presence of thediagnostic HincII site. Blank, not tested. ^(e)Results of directsequencing of the entire gH and gL ORFs (except for a highly GC-rich24-nucleotide portion of gH) or *, a portion of the gH locus containingpositions 753 and 778. Amino acid substitutions are indicated. wt, nosubstitution. Blank, not tested.

Since the majority of the virus isolates had no gD mutations, the gBORFs of one gD:wt (No. 2) were compared with one gD:A185T isolate (No.32). Direct sequencing revealed that both isolates had acquired an S668Nsubstitution creating a new HincII recognition site in the gB gene. Anadditional 28 isolates (Nos. 1, 3-15, 31, 33-44, 46) were screened byHincII-digestion of PCR amplicons spanning the mutant position showingthat 21 of these (Nos. 4-15, 37-44, 46) contained the new HincIIrecognition site. The complete gB ORFs of two of the sevenHincII-negative isolates were sequenced, one harboring gD:A185T (No. 31)and the other containing wild-type gD (No. 1). No amino acidsubstitutions were found in either one. Thus, while 23 out of 30isolates had acquired the gB: S668N substitution, suggesting a role forthis mutation in the new phenotype of these isolates, at least one wasunchanged in both its gD and gB ORFs.

To identify the change(s) in isolate No. 1 responsible for its abilityto enter and form plaques on J/TMCΔC cells, the ORFs in this isolate forthe two other essential entry glycoproteins, gH and gL, were sequenced.While no mutations were found in the gL ORF, the nearly complete gHsequence revealed two amino-acid substitutions, N753K and A778V; thissequence excludes a highly GC-rich 24-nucleotide portion of gH thatcould not be read (positions 2079-2102 in GenBank accession numberX03896). Isolates with identified gD and/or gB mutations were thenanalyzed to determine whether they contained either or both of these gHsubstitutions. Surprisingly, of 7 sequenced isolates, 6 had the same twosubstitutions (N753K/A778V; Nos. 2-4, 31, 44, 46), while one, harboringboth gD:A185T and gB:S668N, showed an A571T substitution in gH (No. 32).These results, particularly the identification of one isolate (No. 1)that carried the gH:N753K/A778V double mutation as the only change inthe four essential glycoprotein genes, strongly suggested that thisdouble mutation had imparted the ability of a number of the isolates,perhaps the majority, to grow and form plaques on J/TMCΔC cells.

Example 6

This example demonstrates evaluation of entry into cells for recombinantviruses containing the gH:N753K/A778V and gH:S668N substitutions(referred to hereafter as gH:KV and gH:668N, respectively) separately orin combination.

To separate the gB:668N and gH:KV substitutions from potential otherchanges in the original isolates, each mutant allele was transferredinto a wild-type virus background by standard homologous recombinationto obtain recombinants named K-gB:668N and K-gH:KV, respectively. Inaddition, a double-recombinant virus, K-gB:668N-gH:KV, was establishedto identify potential combinatorial effects of the gB:668N and gH:KValleles. Likewise, the double recombinant K-gB:NT-gH:KV was generatedcontaining both the gB:N/T mutant allele described earlier and gH:KV.All of these recombinant viruses were confirmed by DNA sequencingthrough the entire gB, gD, and gH ORFs except for the highly GC-rich24-nucleotide portion of gH mentioned earlier. The recombinants, alongwith wild-type KOS virus and K-gB:N/T described above (referred to hereas K-gB:NT), were propagated simultaneously and titered on Vero cells.

K-gB:668N was established by co-transfection of Vero cells with KΔTviral DNA and plasmid pgB1:5668N, followed by plaque purificationthrough three rounds of limiting dilution on Vero cells. PlasmidpgB1:S668N was created by substitution of an S668N-containing gBfragment amplified on DNA from isolate No. 5 for the correspondingfragment of pgB1, a plasmid containing the gB open reading frame (ORF)and flanking regulatory sequences from K26GFP.

K-gH:KV, K-gB:668N-gH:KV, and K-gB:NT-gH:KV were established in twosteps. First, KΔgH, K-gB:668NΔgH, and K-gB:NTΔgH were established byco-transfection of gH-complementing F6 cells with plasmid pΔgH-EGFP andviral DNA of KOS, K-gB:668N, or K-gB:NT, respectively, and purificationof green plaques on F6 cells. Plasmid pΔgH-EGFP was created by replacingthe sequence of the gH ectodomain and transmembrane region in pgH1:wt, aplasmid that contains the gH ORF and flanking regulatory sequences fromKOS, with the EGFP ORF from pEGFP-C1 (Clontech). K-gH:KV,K-gB:668N-gH:KV, and K-gB:NT-gH:KV were then established byco-transfection of Vero cells with plasmid pgH1:N753K/A778V and viralDNA of KΔgH, K-gB:668NΔgH, or K-gB:NTΔgH, respectively, and plaquepurification on Vero cells. Plasmid pgH1:N753K/A778V was created bysubstitution of an N753K/A778V-containing gH fragment amplified on DNAfrom virus isolate No. 1 for the corresponding fragment of pgH1:wt.

K-gH:KVΔgD, K-gB:668N-gH:KVΔgD, and K-gB:NT-gH:KVΔgD were produced byco-transfection of gD-complementingVD60 cells with plasmid pΔgD-EGFP andviral DNA of K-gH:KV, K-gB:668N-gH:KV, or K-gB:NT-gH:KV, respectively,followed by purification of green plaques on VD60 cells.

All recombinant viruses were confirmed by PCR and DNA sequencing throughthe relevant glycoprotein genes or deletions.

Entry assays were performed as described above for K-gB:NT.

As shown in Examples 2-3 above, K-gB:NT has the ability to enter CHO-K1cells, a cell line like J1.1-2 and B78H1 that is resistant to HSV-1 dueto the absence of gD receptors. Recombinant viruses harboring gB:668N orgH:KV were assayed to determine whether they shared this ability. Entryof K-gB:NT into CHO-K1 cells was detectable at an MOI of 3 or higher,whereas no entry was seen by wild-type KOS at an MOI of 30 and onlylimited entry at an MOI of 300. Both K-gB:668N and K-gH:KV reproduciblyshowed somewhat more entry than wild-type KOS, with K-gH:KV reaching alevel that was approximately 10-fold below that of K-gB:NT. Thedouble-recombinant K-gB:668N-gH:KV yielded at least 10-fold more entrythan K-gB:668N or K-gH:KV, comparable to the level observed for K-gB:NT.Moreover, entry by the second double-recombinant virus, K-gB:NT-gH:KV,exceeded that not only of K-gH:KV, but also that of K-gB:NT.Importantly, all of the mutant viruses entered Vero cells to similardegrees as wild-type KOS, excluding the possibility that the observeddifferences in entry into CHO-K1 cells were due to viral inputdifferences. Similar trends were recorded on gD-receptor-deficient B78H1cells. Together, these results indicated that the gB:668N and gH:KValleles possessed a limited ability to compensate for the absence ofauthentic gD receptors in virus entry and that gH:KV could actcooperatively with both gB:668N and gB:NT to enhance this ability.

As shown earlier, gB:N/T entry into gD-receptor-deficient CHO-K1 cellsstill requires gD. To determine if combinations of mutant gB and gHcould eliminate the gD dependence of HSV entry, gD-null viruses wereprepared from K-gB:668N-gH:KV and K-gB:NT-gH:KV, designatedK-gB:668N-gH:KVΔgD and K-gB:NT-gH:KVΔgD, respectively, by replacement ofthe complete gD ORFs with that of GFP. Virus stocks were prepared bypassage through Vero cells to remove the complementing wild-type gDprotein provided by VD60 cells. Since gD-null viruses cannot be titeredon Vero cells, qPCR was used to determine the genome titers [genomecopies (gc)/ml] of these viruses and their gD±counterparts. CHO-K1 cellswere then inoculated with each of the viruses at 300 gc/cell and entrywas visualized at 8 hpi by immunostaining for ICP4. No entry wasobserved with either of the gD-null recombinants, demonstrating that gDis indispensable for CHO-K1 infection even when the viruses carrycombinations of entry-enhancing gB and gH alleles.

These results show that the gB and gH mutations, separately or together,facilitate gD-dependent virus entry into cells that lack authentic gDreceptors. The mechanism may involve gD recognition of minor or crypticgD receptor(s) expressed on the cells, such as nectin-3.

Example 7

This example demonstrates rate of entry analyses for the K-gH:KV andK-gB:NT-gH:KV recombinant viruses compared to wild-type HSV-1 KOS.

As described at Example 3 above, gB:NT accelerates HSV entry into gDreceptor-positive Vero cells. To determine whether the K-gH:KV mutationhad a similar effect, Vero cells were incubated for 30 min at 4° C. withwild-type KOS or gD±mutant viruses at 500 pfu per well, washedthoroughly, incubated at 37 or 30° C. for 0-60 min, and extracellularvirus was inactivated by low-pH treatment. The cells were then overlaidwith methylcellulose-containing media and incubated at 37° C. for 2 daysto allow plaque formation. Percent entry was calculated for each virusrelative to the number of plaques produced by the same virus afterinfection for 2 h at 37° C., conditions that yielded approximately equalplaque numbers for all of the viruses. As can be seen in FIG. 3A-B, thedouble-recombinant K-gB:NT-gH:KV had the fastest entry kinetics at bothtemperatures, followed by K-gB:NT. At 37° C., KOS showed the slowestrate of entry, but at 30° C., no difference was observed between thedramatically reduced entry rates of KOS and K-gH:KV. In additionalexperiments, the entry kinetics of K-gB:668N was found to be similar tothat of K-gH:KV at both temperatures. These observations paralleled thedifferences in entry seen on receptor-deficient cells, suggesting thatthose differences were also due to allele-specific increases in entryrates relative to KOS. It is evident that the absence of authentic gDreceptors causes a general reduction in entry kinetics, and thusdifferences in the entry rates between the viruses affect the totallevels of penetration on receptor-deficient cells for an extended periodof time (e.g., 6 h as in the entry assays of Example 6) compared to Verocells (about 1 h, FIG. 3A).

Together, these data support the likelihood that accelerated entrymediated by the new gB and gH alleles played a role in the selection ofthe original isolates on J/TMCΔC cells.

Example 8

This example demonstrates evaluation of lateral, or (cell-to-cell),virus spread into cells.

To analyze the role of gH mutations in lateral virus spread, donor cells(Vero or VD60) were infected at an MOI of 10 at 37° C. for 2 h toachieve infection of all cells. Viruses used in this experiment wereKOS, K-gB:668N, K-gB:NT, K-gH:KV, K-gB:668N-gH:KV, and K-gB:NT-gH:KV.Extracellular virus was then inactivated by acidic wash and the cellswere incubated at 37° C. for 1 h, trypsinized, and suspended in freshculture media. Equal numbers of infected (donor) cells were seeded ontomonolayers of uninfected (acceptor) cells in a 48-well plate. After a3-h incubation at 37° C., the cells were overlaid withmethylcellulose-containing medium. Two or three days later, the overlaywas removed, and the cells were fixed with 100% methanol andimmunostained with monoclonal mouse anti-VP16 antibody (1:400) (SantaCruz) and Cy3-conjugated sheep anti-mouse IgG (1:400).

Each of the viruses formed plaques on acceptor Vero cells andgD-receptor-transduced B78 cell lines (B78/A, B78/C), regardless of thenature of the gB and gH alleles, although the plaques formed by thethree gH:KV-harboring viruses tended to be somewhat larger than theplaques formed by the other viruses. Surprisingly, however, the threeviruses harboring gH:KV formed plaques on gD-receptor-negative B78 cellsas well (B78/0G, containing a GFP gene controlled by the virus-inducibleICP0 promoter); observation of green fluorescence confirmed that theseplaques consisted of infected acceptor cells rather than donor cells.Analysis at higher magnification showed very small green foci in theKOS, K-gB:668N, and K-gB:NT wells, consistent with a single round ofvirus spread from individual donor cells to their nearest neighborswithout subsequent spread between acceptor cells. In contrast, plaquesformed by the three gH:KV-harboring viruses showed several layers offluorescent cells around a vacant space in the middle, indicative ofinitial virus spread from a central infected Vero cell to its immediateneighbors, followed by multiple rounds of spread from onegD-receptor-negative cell to the next. Similar results were obtainedusing CHO/0G cells, another gD-receptor-deficient line that expressesGFP in response to virus infection. These observations providedcompelling evidence that the gH:KV double mutation enables spreadbetween cells that lack authentic gD receptors. Since neither of the gBmutant alleles displayed a similar ability, this conclusion impliesmechanistic differences and differential roles of gB and gH in entry offree virus and entry by cell-to-cell spread.

Next, to determine the requirement for gD in spread of the differentgH:KV recombinant viruses on gD-receptor-deficient cells, gD-knockoutversions of these viruses were assayed. Infectious center assays wereperformed as above except that the virus stocks were prepared ongD-complementing VD60 cells and VD60 cells were used as donor cells;plaque formation was recorded after 3 days. VP16 immunostaining showedthat the gD-null viruses yielded only single-cell infections or verysmall foci on gD-receptor-deficient B78H1 cells or receptor-positiveVero cells, indicating that gD is required for cell-to-cell spreadregardless of the presence or absence of gD receptors and spread- orentry-enhancing gB/gH mutant alleles in the viruses.

In view of the evidence above that gB:NT, gH:KV, and gB:668N, alone orin combinations, do not complement gD-null viruses in entry, theseresults demonstrate that the different mutant alleles do not confer anew gD-independent mechanism of virus entry from either the media orneighboring cells. Instead, each of these alleles likely acts byamplifying a weak signal from gD, resulting in effective execution ofthe fusion reaction.

Example 9

This example demonstrates evaluation of the effect of gH:KV mutant geneon virus replication and egress.

To examine the possibility that increased virus replication, virionassembly or transport to the cytoplasmic membrane played a role in theincreased lateral spread of K-gH:KV and/or the original selection ofgH:KV-bearing viruses on J/TMCΔC cells, the replication and egressefficiency of K-gH:KV were compared with that of KOS. Vero cells wereinfected for 1 h at an MOI of 3, extracellular virions were inactivatedby acidic wash, and viral titers in cell lysates and media at 4, 8, and24 hpi were determined separately. As shown in FIG. 4A, the two virusesexhibited similar titers in both compartments at each of the selectedtime points. Similar results on B78/C cells (FIG. 4B) indicated thatthis outcome was not dependent on a specific cellular background. Theseobservations argued that the spread-enhancing activity of gH:KV couldnot be attributed to increases in the efficiency of virus replication oregress.

Transfection of HSV-susceptible cells with the four essentialglycoproteins, gB, gD, gH, and gL, causes cell-cell fusion, which isbelieved to reflect, at least in part, the normal functions of theseglycoproteins and their receptors in HSV entry and spread (J Virol72:873-5, 1998; J Gen Virol 81:2017-27, 2000; Virology 279:313-24,2001). To examine the ability of gH:KV in combination with gB, gD, andgL to induce fusion of gD-receptor-deficient B78H1 cells, B78H1 cellswere co-transfected with expression plasmids for gB (pCAgB:wt), gD(pPEP99), gLpPEP101), and either gH:wt (pPEP100 or gH:KV (pCΔgH:KV),using Lipofectamine2000 (Invitrogen). Transfection of B78H1 cells withthe four wild-type genes did not produce detectable cell fusion, inagreement with previous results demonstrating that a gD receptor isessential for HSV glycoprotein-mediated cell fusion (Virology279:313-24, 2001). In contrast, replacement of the wild-type gH plasmidwith the gH:KV version yielded readily detectable multinucleated cellsindicative of cell-cell fusion. No syncytia were observed ontransfection of the same cells with plasmids expressing gB:NT and wildtype gD, gH, and gL.

These results indicate that gH:KV, but not gB:NT, facilitates arate-limiting step in the fusion cascade leading to viral cell-to-cellspread. Together, the distinct properties of gB:NT and gH:KV indicatethat these two alleles address separate rate-limiting steps controllingvirus entry and lateral spread, respectively.

Example 10

This example demonstrates that hyperactive glycoprotein B mutationsaugment fully retargeted HSV infection.

To retarget virus entry exclusively to epidermal growth factor receptor(EGFR)-bearing cells, gD residues essential for binding to the naturalreceptors were mutated or deleted, and EGF or an EGFR-specificsingle-chain antibody (scFv) were inserted near the amino terminus.

FIG. 5 illustrates modifications in the gD coding sequence designed todetarget HSV from its natural entry receptors and retarget the virus toEGFR. Detargeting mutations included a small (residues 7-11) or largerdeletion (residues 2-24) in the HVEM-binding N-terminal region and oneor two amino-acid substitutions (Y38C or A3C/Y38C) to ablate virus entrythrough nectin-1. To retarget these constructs, an scFv directed againstEGFR (scEGFR) was inserted in the 2-24 deletion or, as described inExample 4, between residues 24 and 25. In addition, constructscontaining the EGF sequence at the same positions were also created.

To assess the complementing activity and specificity of the retargetedgD constructs of FIG. 5 (designated gD:Δ224/38C-EGF, gD:Δ224/38C-scEGFR,gD:3C/Δ711/38C-EGF, and gD:3C/Δ711/38C-scEGFR), transientcomplementation assays were performed using gD-null viruses thatexpressed either wild-type gB or the gB:NT mutant allele (K-gB:wtΔgD andK-gB:N/TΔgD described in Example 4). Vero cells were transfected withexpression plasmids for the parental detargeted gD genes (gD:Δ224/38Cand gD:3C/Δ711/38C, FIG. 5) or the retargeted gD genes and the cellswere infected the next day with K-gB:wtΔgD or K-gB:N/TΔgD. Supernatantswere harvested the following day and used to infect HSV-resistant babyhamster kidney J1.1-2 cells or J1.1-2 derivatives expressing HVEM (J/A),nectin-1 (J/C), or EGFR (J/EGFR). Results showed that the gD genesharboring the EGFR targeting sequences (EGF or scEGFR) allowed entry ofthe gB:NT/gD-null virus into J/EGFR cells, but entry of thegB:wt/gD-null virus into these cells was essentially undetectable. Inaddition, none of the mutant gD constructs allowed entry of either virusinto J1.1-2, J/A, or J/C cells whereas the wt gD gene enabled entry ofboth viruses into J/A and J/C cells.

The same transiently complemented viruses were also tested on Vero cellscommonly used for HSV propagation. These cells express simian EGFRendogenously as well as natural gD receptors. Similar to J/EGFR cells,both the EGF- and the scEGFR-retargeted gD genes allowed gB:NT-dependentgD-null virus entry into Vero cells, while no entry was observed in theabsence of the EGFR-targeting ligands or by complemented gB:wt/gD-nullvirus. Unlike J/EGFR cells, however, Vero cells were also susceptible toboth viruses complemented with gD:wt, allowing a direct comparison ofthe relative levels of normal and retargeted virus entry via theirrespective receptors. This comparison indicated that entry of thegB:NT/gD-null virus complemented with either of the two scEGFR-harboringgD alleles was almost as efficient as entry by gD:wt-complementedgD-null/gB:wt virus.

The scEGFR sequence was also inserted into a deletion of residues 61through 218 in gD (gD:A61-218-scEGFR), a position used by Menotti andcolleagues for the insertion of an anti-HER-2 scFv resulting inefficient virus retargeting to HER-2-expressing cells. Complementationof gB:NT/gD-null or gB:wt/gD-null viruses with the gD:A61-218-scEGFRconstruct did not yield detectable entry into J/EGFR cells or CHO/EGFRcells, another EGFR-transduced HSV-resistant cell line. Likewise, noentry was observed with EGF inserted into the 61-218 deletion.

Together, these results indicate that (i) the detargeting mutations inboth types of constructs (gD:Δ224/38C and gD:3C/Δ711/38C) wereeffective; (ii) targeting was accomplished not only by scEGFR, but alsoby the natural receptor ligand, EGF; and (iii) the gB:NT allele raisedthe efficiency of retargeted infection to a level near that seen with acomparable wt virus entering via the natural HSV receptors. Further, theresults imply that the 61-218-deleted version of gD does not represent auniversally effective platform for gD retargeting by ligand insertion.

Example 11

This example demonstrates efficiency, specificity, and kinetics ofretargeted HSV entry.

The scEGFR-containing gD constructs were used to establish retargetedgB:wt and gB:NT recombinant viruses by homologous recombination with thecorresponding gD-null viruses (K-gB:wtΔgD and K-gB:N/TΔgD) ongD-complementing VD60 cells. Recombinants were plaque-purified bymultiple rounds of limiting dilution and finally passaged throughnon-complementing Vero cells to obtain virus preparations free ofwild-type gD protein. Absolute virus titers expressed as genome copies(gc)/ml were determined by qPCR for the viral immediate early geneICP47; these titers allow comparison of the entry efficiencies betweenviruses that differ in their recognition of entry receptors.

Fluorescence analysis showed that gB:NT recombinant viruses expressingeither of the scEGFR-retargeted gD constructs entered J/EGFR cellsefficiently, comparable to the entry of parental wt HSV-1 strain KOSinto J/A or J/C cells that express authentic HSV receptors. In contrast,the gB:wt versions of these recombinants entered J/EGFR cells some100-fold less efficiently. None of the retargeted viruses detectablyentered into J1.1-2, J/A, or J/C cells even at high virus input (1,000gc/cell), demonstrating that the detargeting mutations in the retargetedgD constructs were effective in abolishing virus entry via the naturalHSV receptors. Similar results were obtained on CHO-K 1 cells andreceptor-expressing derivatives. Consistent with these results, thegB:NT version of each of the scEGFR-retargeted viruses entered Verocells as efficiently as wt KOS virus while the gB:wt versions showedlimited entry only at high virus concentrations. Entry of the gB:NTversion of the gD:Δ224/38C-scEGFR virus into Vero cells was confirmed tobe EGFR dependent by pretreating the cells with anti-EGFR monoclonalantibody (mAb), resulting in a dose-dependent inhibition of entrywithout effect on entry of wt KOS virus

As described above, the gB:NT allele accelerates HSV entry via normaland cryptic receptors, and thus it was of interest to compare the rateof entry of the gB:NT and gB:wt versions of the gD:Δ224/38C-scEGFRvirus. Briefly, Vero-derived gD-complementing VD60 cells were infectedfor 0-60 min, extracellular virus was removed, and plaques were counted2d later; the use of VD60 cells eliminates differences in gD-dependentlateral virus spread following the initial entry of extracellular virusand thus minimizes differences in the rate of plaque formation byintracellular viruses expressing different gD genes. Wild-type KOSentered VD60 cells gradually over 60 min while the gB:wt version of theretargeted virus entered at a highly reduced rate (FIG. 6). However, theretargeted virus expressing the gB:NT allele entered VD60 cells morerapidly not only than the gB:wt version, but also than wt KOS virus,reaching a plateau value at approximately 20 min.

These results demonstrate that the gB:NT allele dramatically increasesthe kinetics of retargeted virus entry via a target receptor that bearsno relationship to any known common or cryptic HSV receptors.

Example 12

This example demonstrates retargeted virus infection and killing ofhuman tumor lines.

Entry and cell-killing abilities of the gD:Δ224/38C-scEGFR viruses andKOS were compared on a panel of HSV-permissive human tumor lines knownto express EGFR, including lung carcinoma A549, colon carcinoma HT29,pancreatic carcinoma BxPC3, glioblastomas U87 and SNB19, and epidermoidcarcinoma A431. Inoculation at 10 gc/cell yielded readily detectableentry of the gB:NT version of the retargeted virus, but only minimalentry of the gB:wt version. KOS virus entered each of these cell linesat a similar efficiency as the retargeted gB:NT virus, as observedearlier with Vero cells (FIG. 6). To confirm the receptor specificity ofthe retargeted virus on HSV-susceptible human cells, HT29 cells weretreated with anti-EGFR mAb prior to infection. Dose-dependent inhibitionof entry of the retargeted gB:NT virus, but not KOS, was observed.

To assess the oncolytic potential of these retargeted gB:wt and gB:NTviruses, A549 and U87 cells were infected with increasing amounts of theviruses and cell viability was determined by MTT assay at 3 or 6 dayspost-infection. As shown in FIG. 7, the retargeted gB:NT virus showedefficient cell killing at 10 gc/cell, similar to KOS. However, A549cells were killed less efficiently by the retargeted virus than by KOSat lower virus input, an observation that may relate to the efficiencyof lateral spread which can potentially be enhanced by spread-enhancingmutations such as found in the gH:KV allele described above. Asexpected, the retargeted gB:wt virus showed less killing activity onboth cell lines.

These results show that the retargeted gB:NT virus has oncolyticcapabilities comparable to a wild-type virus, although efficiency islikely affected by the targeted cell type.

Example 13

This example demonstrates specificity and oncolytic potency of theretargeted gB:NT virus in vivo.

The EGFR scFv used in retargeted viruses as described herein is specificfor human EGFR while wt HSV-1 KOS is neurotoxic in mouse strains such asBALB/c. Thus, neurotoxicity testing in mice was performed as a stringentmeasure of the specificity of the retargeted gB:NT virus. Groups of fourmice were injected intracranially with 5×10³ gc KOS or a 100,000-foldhigher dose of the retargeted gB:NT virus (5×10⁸ gc). Of the animalsinjected with KOS virus, one died on day 6, two on day 7, and one on day9. In contrast, all four mice injected with the retargeted virusremained alive and symptom-free throughout the 47-day observationperiod. In a separate experiment, brain sections of injected mice wereanalyzed for the presence of virus by immunostaining for the viral ICP4protein. Abundant virus was detected in the brain of a mouse that haddied on day 21 after receiving KOS at a dose of 1×10³ gc, while littlevirus was detected in brain sections from a mouse that had beensacrificed on day 37 (no symptoms) after injection of 5×10⁸ gc of theretargeted gB:NT virus. Virus stocks used for these experiments were thesame as those used in Example 11, showing comparable infection ofHSV-susceptible cells lines expressing human or simian EGFR by equalamounts of KOS and the retargeted virus, strongly arguing against thepossibility that the observed differences in neurotoxicity between thesetwo viruses were due to dosing errors. Thus, the results confirmed thatthe retargeted gB:NT virus was effectively detargeted from its naturalreceptors in mouse brain and was harmless in this complex in vivoenvironment lacking the targeted receptor.

Next, the retargeted gB:NT virus was examined to determine whether itwould preferentially accumulate in EGFR-positive human tumors in nudemice. Following the establishment of subcutaneous U87 flank tumors(700-1,000 mm³), equal gc of KOS and the retargeted gB:NT virus wereadministered by tail-vein injection. The animals were sacrificed 2 dayslater and the amount of virus in the tumor and various organs determinedby qPCR for the viral ICP4? gene. As shown in FIG. 8A, the number of KOSgenomes per 100 ng tissue DNA was lower in the tumors than in the liver,spleen or intestine of the same animals, but comparable to the lownumbers in other organs. In contrast, the retargeted virus was detectedat 100-1,000-fold higher levels or more in the tumors than in othertissues. These results clearly demonstrate that the retargeted viruspreferentially homed to the human tumor tissue.

To examine the anti-tumor efficacy of the retargeted gB:NT virus, U87flank tumors averaging approximately 140 mm³ in size were injected withPBS or virus at 5×10⁸ gc. As shown in FIG. 8B, PBS-injected tumorsincreased in size to 900-1,000 mm³ over a period of 29 days, whereas thegrowth of tumors injected with the retargeted virus was suppressedduring the first 20 days, resulting in only a limited increase in sizeat the end of the observation period (FIG. 8B).

While these results indicate that a single injection of the retargetedvirus was not sufficient for complete tumor eradication, they providecompelling evidence of effective tumoricidal activity without adverseeffects attributable to the virus.

Example 14

This example demonstrates HSV vector targeting to carcinoembryonicantigen (CEA), a cell surface molecule frequently overexpressed in humancancers.

A CEA-retargeted gD gene, gD:Δ224/38C-scCEA, was created by insertingthe coding sequences of the F39 anti-CEA scFv (scCEA, 238 amino acids)into the 2-24 deletion of gD:Δ224/38C (FIG. 9) and was tested fortransient complementation of K-gB:wtΔgD and K-gB:NTΔgD on CHO cellsexpressing CEA or EGFR. The scCEA in gD was found to enable entry of thegB:NTΔgD virus into CHO-CEA, but not CHO-EGFR cells, and no entry intoeither cell line was detected for the complemented gB:wtΔgD virus. Ascontrols, the scEGFR-retargeted gD:Δ224/38C construct efficientlycomplemented K-gB:NTΔgD, but not K-gB:wtΔgD, on CHO-EGFR cells.

Recombinant viruses were then prepared with the CEA-retargeted gD alleleand examined for CEA-dependent entry into CEA-positive MKN45 andCEA-negative MKN74 human gastric carcinoma cells. KOS entered into bothcell lines, consistent with previous observations (Mol. Ther. 19,507-514, 2011), but the retargeted viruses entered only into MKN45cells. More entry into MKN45 cells was observed with the gB:NT versionof the scCEA virus than with the gB:wt version, although the differencewas not as dramatic as that seen with the scEGFR viruses, and entry bythe gB:NT version did not quite reach the level of KOS entry.

These results demonstrate that ligands other than for EGFR can beinserted into gD:Δ224/38C to accomplish efficient infection throughdifferent non-HSV receptors in combination with the gB:NT allele. It isexpected that the efficiency of retargeted infection relative to KOSwill vary with factors such as the receptor/ligand pair, including theabundance and nature of the targeted receptors.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-17. (canceled)
 18. A method of killing a cancerous cell comprisingcontacting the cell with a mutant HSV vector comprising a modifiedglycoprotein B (gB) wherein, when the parental vector is HSV1 K26GFP,the modified gB comprises a substitution at amino acid residues gB:D285and gB:A549 or when the parental vector is a homologous HSV-1 or HSV-2vector, the modified gB comprises a substitution at amino acid residuesgB:D285 and gB:A549 of the homologous HSV vector, wherein the gB:D285residue correlates to X in VYPYXEFVL (SEQ ID NO:1) of HSV1 K26GFP andthe gB:A549 residue correlates to X in KLNPNXIAS (SEQ ID NO:2) of HSV1K26GFP.
 19. The method of claim 18, wherein the substitution at gB:D285is gB:D285N and the substitution at gB:A549 is gB:A549T.
 20. The methodof claim 18, wherein the mutant HSV vector further comprises anexogenous expression cassette.
 21. The method of claim 20, wherein theexpression cassette comprises a target sequence for a cellular microRNA.22. The method of claim 18, wherein the mutant HSV vector furthercomprises a non-native ligand capable of specifically binding a surfacecomponent of a predetermined cell type.
 23. The method of claim 22,wherein the predetermined cell type is a cancer cell.
 24. The method ofclaim 22, wherein the ligand is incorporated into a viral envelopeglycoprotein of the HSV vector.
 25. The method of claim 24, wherein theviral envelope glycoprotein is gD or gC.
 26. The method of claim 22,wherein the component is a protein selected from the group consisting ofEGFR, EGFRvIII, CEA, and ClC-3/annexin-2/MMP-2.
 27. The method of claim1, wherein the cancerous cell is in vivo or in vitro.
 28. An HSV vectorcomprising a modified glycoprotein B (gB) wherein, when the parentalvector is HSV1 K26GFP, the modified gB comprises a substitution at aminoacid residues gB:D285 and gB:A549 or when the parental vector is ahomologous HSV-1 or HSV-2 vector, the modified gB comprises asubstitution at amino acid residues gB:D285 and gB:A549 of thehomologous HSV vector, wherein the gB:D285 residue correlates to X inVYPYXEFVL (SEQ ID NO:1) of HSV1 K26GFP and the gB:A549 residuecorrelates to X in KLNPNXIAS (SEQ ID NO:2) of HSV1 K26GFP.
 29. The HSVvector of claim 28, wherein the substitution at gB:D285 is gB:D285N andthe substitution at gB:A549 is gB:A549T.
 30. The HSV vector of claim 28,further comprising an exogenous expression cassette.
 31. The HSV vectorof claim 30, wherein the expression cassette comprises a target sequencefor a cellular microRNA.
 32. The HSV vector of claim 28, furthercomprising a non-native ligand capable of specifically binding a surfacecomponent of a predetermined cell type.
 33. The HSV vector of claim 32,wherein the predetermined cell type is a cancer cell.
 34. The HSV vectorof claim 32, wherein the ligand is incorporated into a viral envelopeglycoprotein of the HSV vector.
 35. The HSV vector of claim 34, whereinthe viral envelope glycoprotein is gD or gC.
 36. The HSV vector of claim34, wherein the component is a protein selected from the groupconsisting of EGFR, EGFRvIII, CEA, and ClC-3/annexin-2/MMP-2.